How new lesions form in Multiple Sclerosis (MS) is unknown. MS lesions that are less than 24 hours in age are characterized by blood-brain barrier breakdown, oligodendrocyte death, and early swelling of myelin, with a conspicuous absence of infiltrating lymphocytes or macrophages. This constellation of findings supports a toxic or viral etiology for the newly forming lesion rather than an autoimmune etiology. We hypothesize that Clostridium perfringens epsilon toxin drives new lesion formation in MS based on its specificity for CNS endothelial cells and oligodendrocytes. Murrell first proposed the potential link between epsilon toxin and MS in 1986 but sensitive reagents and assays for detection of the toxin were lacking. In this application we test the hypothesis that there is an association between the presence of epsilon toxin, or the epsilon toxin producing strains of Clostridium perfringens, and Multiple Sclerosis. We have generated highly specific monoclonal antibodies directed against epsilon toxin and now have a suite of sensitive assays for detection of the toxin in clinical samples. We have designed a clinical study in which 40 MS and 40 healthy control subjects will be enrolled over a period of 1.5 years. Study subjects will provide blood and fecal samples for analysis. Blood will be studied by flow cytometry for the presence of toxin bound to lymphocytes or endothelial microparticles, and by ELISA for the presence of toxin in plasma. Fecal samples will be cultured in perfringens-selective media under strict anaerobic conditions and supernatants studied for the presence of epsilon toxin by ELISA. In samples (plasma or bacterial culture) where ELISA detects epsilon, a western blot confirmation will be performed. In bacterial cultures where we detect epsilon toxin by ELISA, we will perform PCR-based genotyping of C. perfringens to identify the relevant strains. In addition, epsilon-positive fecal cultures will be further processed to isolate the potentially pathogenic organism through culture on selective agar medium. This project (1) assesses the feasibility of a novel avenue of investigation, and (2) the involves experiments that could lead to a breakthrough in a particular field, thus fitting 2 of the 3 criteria the R21 mechanism is intended to support. We recognize that the underlying hypothesis of this project seemingly goes against the central dogma that MS is an autoimmune disease. In reality, the two are very compatible, since we propose that new lesions formed as a result of epsilon toxin have a defined probability of progressing to the typical ?active? MS lesion characterized by perivenular cuffs of lymphocytes, myelin-laden macrophages, demyelination, and variable degrees of axonal injury.